Method for culturing and assaying cells

ABSTRACT

An improved system for screening a multiple of candidate therapeutic or chemotherapeutic agents for efficacy as to a specific patient, in which a tissue sample from the patient is harvested, cultured and separately exposed to a plurality of treatments and/or therapeutic agents for the purpose of objectively identifying. One particularly important tissue sample preparation technique is the initial preparation of cohesive multicellular particulates of the tissue sample. For assays concerning cancer treatment, a two-stage evaluation is contemplated in which both acute cytotoxic and longer term inhibitory effect of a given anti-cancer agent are investigated. The tissue sample technique of the present invention is also useful in assaying expression and/or secretion of various markers, factors or antigens present on or produced by the cultured cells for diagnostic purposes and for using such expression to monitor the applicability of certain candidate therapeutic or chemotherapeutic agents and the progress of treatment with those agents.

RELATED APPLICATION

This is a continuation-in-part of U.S. application Ser. No. 08/679,056,filed Jul. 12, 1996, now U.S. Pat. No. 5,728,541.

FIELD OF THE INVENTION

The invention relates to screening and testing of active agents,including chemotherapeutic agents, to predict potential efficacy inindividual patients in whom treatment with such agents is indicated. Theinvention also relates to a method for screening for expression ofcellular markers, secreted factors or tumor antigens by cells fordetermining the disease state of the cells and for monitoring thepotential efficacy of treatment agents.

INTRODUCTION

All active agents including chemotherapeutic active agents are subjectedto rigorous testing as to efficacy and safety prior to approval formedical use in the United States. Methods of assessing efficacy haveincluded elaborate investigations of large populations in double blindstudies as to a given treatment method and/or active agent, withconcomitant statistical interpretation of the resulting data, but theseconclusions are inevitably generalized as to patient populations takenas a whole. In many pharmaceutical disciplines and particularly in thearea of chemotherapy, however, the results of individual patient therapymay not comport with generalized data—to the detriment of the individualpatient. The need has been long recognized for a method of assessing thetherapeutic potential of active agents, including but not limited tochemotherapeutic agents, for their efficacy as to a given individualpatient, prior to the treatment of that patient.

Prior art assays already exist which expose malignant tissue of varioustypes to a plurality of active agents, for the purpose of assessing thebest choice for therapeutic administration. For example, in Kruczynski,A., et al., “Evidence of a direct relationship between the increase inthe in vitro passage number of human non-small-cell-lung cancerprimocultures and their chemosensitivity,” Anticancer Research, vol. 13,no. 2, pp. 507-513 (1993), chemosensitivity of non-small-cell-lungcancers was investigated in in vivo grafts, in in vitro primoculturesand in commercially available long-term cancer cell lines. The increasein chemosensitivity was documented and correlated with morphologicalchanges in the cells in question. Sometimes animal model malignant cellsand/or established cell cultures are tested with prospective therapyagents, see for example Arnold, J. T., “Evaluation of chemopreventiveagents in different mechanistic classes using a rat tracheal epithelialcell culture transformation assay,” Cancer Res., vol. 55, no. 3, pp.537-543 (1995).

In vitro prior art techniques present the further shortcoming thatassayed cells do not necessarily express the cellular markers they wouldexpress in vivo. This is regrettable because the determination ofexpression of certain secreted or cellular markers, secreted factors ortumor antigens or lack thereof can be useful for both identification andtherapeutic purposes. For instance, members of the fibrinolytic systemsuch as urokinase-type plasminogen activator (u-PA) and plasminogenactivator inhibitors type 1 (PAI-1) are up-regulated in malignant braintumors. See, e.g., Jasti S. Rao, et al., “The Fibrinolytic System inHuman Brain Tumors: Association with Pathophysiological Conditions ofMalignant Brain Tumors,” Advances in Neuro-Oncology II, Kornblith PL,Walker Md. (eds) Futura 1997. Other secreted factors such asα-fetoprotein, carcinoembryonic antigen and transforming growth factorsα and β have been found to be indicative of various cancers and/orcancer progression (see also, Singhal et al., “Elevated PlasmaOsteopontin in Metastatic Breast Cancer Associated with Increased TumorBurden and Decreased Survival,” Clinical Cancer Research, Vol. 3,605-611, April 1997; Kohno et al., “Comparative Studies of CAM 123-6 andCarcinoembryonic Antigen for the Serological Detection of PulmonaryAdenocarcinoma,” Cancer Detection and Prevention, 21(2): 124-128(1997)). These examples are but a few of the many factors that may beused to identify diseased cells.

Often the diseased cells express a cellular marker that is indicative ofa certain disease state or lack thereof. However, one aspect of theculture techniques of the present invention is that the cultureddiseased cells do not necessarily have to be the cells expressing thefactor to be assayed. One question that inevitably arises whenconsidering whether a serum marker is indicative of a particular cancercell is, which cells produce the marker, the cell or the tissue in whichthe cancer cells grow? See e.g. Singhal et al., p 610. By co-culturingthe cancerous tissue within a multicellular particulate of itsoriginating tissue, the cells (both the diseased cells or thesurrounding cells) are better able to retain their production ofcharacteristic markers.

When actual patient cells are used to form in vitro assays focused onindividual patients, in typical prior art processes the cells areharvested (biopsied) and trypsinized (connective tissue digested withthe enzyme trypsin) to yield a cell suspension suitable for conversionto the desired tissue culture form. The in vitro tissue culture cellcollections which result from these techniques are generally plagued bytheir inability accurately to imitate the chemosensitivity of theoriginal tumor or other cell biopsy. These collections often do notexpress cellular markers in the same manner that they would in vivo.Standard cloning and tissue culture techniques are moreover excessivelycomplicated and expensive for use in a patient-by-patient assay setting.A need thus remains for a technique of tissue culture preparation whichprovides cell cultures, for drug screening purposes, in which aftersimple preparation the cell cultures react in a manner equivalent totheir in vivo reactivity, to enable drug or chemotherapeutic agentscreening as to a particular patient for whom such screening isindicated. A need also remains for a technique of tissue culturepreparation which provides cell cultures for screening for expressedmarkers or factors where the cultured cells express the markers orfactors in a manner indicative of their in vivo expression of the same.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is an improvedsystem for screening a multiple of candidate therapeutic orchemotherapeutic agents for efficacy as to a specific patient, in whicha tissue sample from the patient is harvested, cultured and separatelyexposed to a plurality of treatments and/or therapeutic agents for thepurpose of objectively identifying the best treatment for the culturedcells obtained from the patient. The culture techniques of the presentinvention also result in a monolayer of cells that express cellularmarkers, secreted factors and tumor antigens in a manner representativeof their expression in vivo. Specific method innovations such as tissuesample preparation techniques render this method practically as well astheoretically useful. One particularly important tissue samplepreparation technique is the initial preparation of cohesivemulticellular particulates of the tissue sample, rather thanenzymatically dissociated cell suspensions or preparations, for initialtissue culture monolayer preparation. With respect to the culturing ofmalignant cells, for example, it is believed (without any intention ofbeing bound by the theory) that by maintaining the malignant cellswithin a multicellular particulate of the originating tissue, growth ofthe malignant cells themselves is facilitated versus the overgrowth offibroblasts or other cells which tends to occur when suspended tumorcells are grown in culture. Practical monolayers of cells may thus beformed to enable meaningful screening of a plurality of treatmentsand/or agents as well as meaningful identification of cellular markers.In the drug assays, growth of cells is monitored to ascertain the timeto initiate the assay and to determine the growth rate of the culturedcells; sequence and timing of drug addition is also monitored andoptimized. By subjecting uniform samples of cells to a wide variety ofactive agents (and concentrations thereof), the most efficacious agentcan be determined. For assays concerning cancer treatment, a two-stageevaluation is contemplated in which both acute cytotoxic and longer terminhibitory effects of a given anti-cancer agent are investigated.

With regard to the identification of expressed cellular markers,secreted factors or tumor antigens, with the initial culturing of themulticellular particulates it is believed (without any intention ofbeing bound by the theory) that because the cells are grown underconditions closer to those found in vivo, the cells express theircellular markers, secreted factors and tumor antigens in a manner moreclosely resembling their expression in vivo.

By assaying the culture media obtained from growing a monolayeraccording to the inventive method or by histochemically and/orimmunohistochemically assaying the cells grown under such conditions, amore accurate profile of the cellular markers or factors is obtained.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a system for screening a multiple of candidatetherapeutic or chemotherapeutic agents for efficacy as to a specificpatient, in which a tissue sample from the patient is harvested andseparately exposed to a plurality of treatments and/or therapeuticagents for the purpose of objectively identifying the best treatment oragent. Specific method innovations such as tissue sample preparationtechniques render this method practically as well as theoreticallyuseful. One particularly important tissue sample preparation techniqueis the initial preparation of cohesive multicellular particulates(explants) of the tissue sample, rather than enzymatically dissociatedcell suspensions or preparations, for initial tissue culture monolayerpreparation. Cell growth, and sequence and timing of drug addition, aremonitored and optimized.

An important application of the present invention is the screening ofchemotherapeutic agents and other antineoplastic therapies againsttissue culture preparations of malignant cells from the patients fromwhom malignant samples are biopsied. Related anti-cancer therapies whichcan be screened using the inventive system are both radiation therapyand agents which enhance the cytotoxicity of radiation, as well asimmunotherapeutic anti-cancer agents. Screening processes for treatmentsor therapeutic agents for nonmalignant syndromes are also embracedwithin this invention, however, and include without limitation agentswhich combat hyper proliferative syndromes, such as psoriasis, or woundhealing agents. Nor is the present efficacy assay limited only to thescreening of active agents which speed up (healing) or slow down(anti-cancer, anti-hyper proliferative) cell growth because agentsintended to enhance or to subdue intracellular biochemical functions maybe tested in the present tissue culture system also. For example, theformation or blocking of enzymes, neurotransmitters and otherbiochemicals may be screened with the present assay methods prior totreatment of the patient.

When the patient is to be treated for the presence of tumor, in thepreferred embodiment of the present invention a tumor biopsy of >100 mgof non-necrotic, non-contaminated tissue is harvested from the patientby any suitable biopsy or surgical procedure known in the art. Biopsysample preparation generally proceeds as follows under a Laminar FlowHood which should be turned on at least 20 minutes before use. Reagentgrade ethanol is used to wipe down the surface of the hood prior tobeginning the sample preparation. The tumor is then removed, understerile conditions, from the shipping container and is minced withsterile scissors. If the specimen arrives already minced, the individualtumor pieces should be divided into four groups. Using sterile forceps,each undivided tissue quarter is then placed in 3 ml sterile growthmedium (Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin) and systematically minced byusing two sterile scalpels in a scissor-like motion, or mechanicallyequivalent manual or automated opposing incisor blades. Thiscross-cutting motion is important because the technique creates smoothcut edges on the resulting tumor multicellular particulates. Preferablybut not necessarily, the tumor particulates each measure 1 mm³. Aftereach tumor quarter has been minced, the particles are plated in cultureflasks using sterile pasteur pipettes (9 explants per to-25 or 20particulates per to-75 flask). Each flask is then labeled with thepatient's code, the date of explanation and any other distinguishingdata. The explants should be evenly distributed across the bottomsurface of the flask, with initial inverted incubation in a 37° C.incubator for 5-10 minutes, followed by addition of about 5-10 mlsterile growth medium and further incubation in the normal, non-invertedposition. Flasks are placed in a 35° C., non-CO₂ incubator. Flasksshould be checked daily for growth and contamination. Over a period of afew weeks, with weekly removal and replacement of 5 ml of growth medium,the explants will foster growth of cells into a monolayer. With respectto the culturing of malignant cells, it is believed (without anyintention of being bound by the theory) that by maintaining themalignant cells within a multicellular particulate of the originatingtissue, growth of the malignant cells themselves is facilitated versusthe overgrowth of fibroblasts (or other unwanted cells) which tends tooccur when suspended tumor cells are grown in culture.

The use of the above procedure to form a cell monolayer culturemaximizes the growth of malignant cells from the tissue sample, and thusoptimizes ensuing tissue culture assay of chemotherapeutic action ofvarious agents to be tested. Enhanced growth of actual malignant cellsis only one aspect of the present invention, however; another importantfeature is the growth rate monitoring system used to oversee growth ofthe monolayer once formed. Once a primary culture and its derivedsecondary monolayer tissue culture has been initiated, the growth of thecells is monitored to ascertain the time to initiate the chemotherapyassay and to determine the growth rate of the cultured cells.

Monitoring of the growth of cells is conducted by counting the cells inthe monolayer on a periodic basis, without killing or staining the cellsand without removing any cells from the culture flask. The counting maybe done visually or by automated methods, either with or without the useof estimating techniques known in the art (counting in a representativearea of a grid multiplied by number of grid areas, for example). Datafrom periodic counting is then used to determine growth rates which mayor may not be considered parallel to growth rates of the same cells invivo in the patient. If growth rate cycles can be documented, forexample, then dosing of certain active agents can be customized for thepatient. The same growth rate can be used to evaluate radiationtreatment periodicity, as well. It should be noted that with the growthrate determinations conducted while the monolayers grow in their flasks,the present method requires no hemocytometry, flow cytometry or use ofmicroscope slides and staining, with all their concomitant labor andcost.

Protocols for monolayer growth rate generally use a phase-contrastinverted microscope to examine culture flasks incubated in a 37° C. (5%CO₂) incubator. When the flask is placed under the phase-contrastinverted microscope, ten fields (areas on a grid inherent to the flask)are examined using the 10× objective, with the proviso that the tenfields should be non-contiguous, or significantly removed from oneanother, so that the ten fields are a representative sampling of thewhole flask. Percentage cell occupancy for each field examined is noted,and averaging of these percentages then provides an estimate of overallpercent confluency in the cell culture. When patient samples have beendivided between two or among three or more flasks, an average cell countfor the total patient sample should be calculated. The calculatedaverage percent confluency should be entered into a process log toenable compilation of data—and plotting of growth curves—over time.Monolayer cultures may be photographed to document cell morphology andculture growth patterns. The applicable formula is:${{Percent}\quad{confluency}} = {\frac{{estimate}\quad{of}\quad{the}\quad{area}\quad{occupied}\quad{by}\quad{cells}}{{total}\quad{area}\quad{in}\quad{an}\quad{observed}\quad{field}}.}$As an example, therefore, if the estimate of area occupied by the cellsis 30% and the total area of the field is 100%, percent confluency is30/100, or 30.

Adaptation of the above protocol for non-tumor cells is straightforwardand generally constitutes an equivalent procedure.

Active agent screening using the cultured cells does not proceed in theinitial incubation flask, but generally proceeds using plates such asmicrotiter plates. The performance of the chemosensitivity assay usedfor screening purposes depends on the ability to deliver a reproduciblecell number to each row in a plate and/or a series of plates, as well asthe ability to achieve an even distribution of cells throughout a givenwell. The following procedure assures that cells are reproduciblytransferred from flask to microtiter plates, and cells are evenlydistributed across the surface of each well.

The first step in preparing the microtiter plates is, of course,preparing and monitoring the monolayer as described above. The followingprotocol is exemplary and susceptible of variation as will be apparentto one skilled in the art. Cells are removed from the culture flask anda cell pellet is prepared by centrifugation. The cell pellet derivedfrom the monolayer is then suspended in 5 ml of the growth medium andmixed in a conical tube with a vortex for 6 to 10 seconds. The tube isthen rocked back and forth 10 times. A 36 μl droplet from the center ofthe conical tube is pipetted onto one well of a 96 well plate. A freshpipette is then used to pipette a 36 μl aliquot of trypan blue solution,which is added to the same well, and the two droplets are mixed withrepeated pipette aspiration. The resulting admixture is then dividedbetween two hemocytometer chambers for examination using a standardlight microscope. Cells are counted in two out of four hemocytometerquadrants, under 10× magnification. Only those cells which have nottaken up the trypan blue dye are counted. This process is repeated forthe second counting chamber. An average cell count per chamber is thusdetermined. Using means known in the art, the quadrant count values arechecked, logged, multiplied by 10⁴ to give cells/ml, and the totalamount of fluid (growth medium) necessary to suspend remaining cellaliquots is calculated accordingly.

After the desired concentration of cells in medium has been determined,additional cell aliquots from the monolayer are suspended in growthmedium via vortex and rocking and loaded into a Terasaki dispenser knownin the art. Aliquots of the prepared cell suspension are delivered intothe microtiter plates using Terasaki dispenser techniques known in theart. A plurality of plates may be prepared from a single cell suspensionas needed. Plates are then wrapped in sterile wet cotton gauze andincubated in an incubator box by means known in the art.

After the microtiter plates have been prepared, exposure of the cellstherein to active agent is conducted according to the followingexemplary protocol. During this portion of the inventive assay, theappropriate amount of specific active agent is transferred into themicrotiter plates prepared as described above. A general protocol, whichmay be adapted, follows. Each microtiter plate is unwrapped from its wetcotton gauze sponge and microscopically examined for cell adhesion.Control solution is dispensed into delineated rows of wells within thegrid in the microtiter plate, and appropriate aliquots of active agentto be tested are added to the remaining wells in the remaining rows.Ordinarily, sequentially increasing concentrations of the active agentbeing tested are administered into progressively higher numbered rows inthe plate. The plates are then rewrapped in their gauze and incubated inan incubator box at 37° C. under 5% CO₂. After a predefined exposuretime, the plates are unwrapped, blotted with sterile gauze to remove theagent, washed with Hank's Balance Salt Solution, flooded with growthmedium, and replaced in the incubator in an incubator box for apredefined time period, after which the plates may be fixed and stainedfor evaluation.

Fixing and staining may be conducted according to a number of suitableprocedures; the following is representative. After removal of the platesfrom the incubator box, culture medium is poured off and the plates areflooded with Hank's Balance Salt Solution. After repeated flooding (withagitation each time) the plates are then flooded with reagent gradeethanol for 2-5 minutes. The ethanol is then poured off. Staining isaccomplished with approximately 5 ml of Giemsa Stain per plate, althoughvolume is not critical and flooding is the goal. Giemsa stain should beleft in place 5 min. ±30 seconds as timing influences stainingintensity. The Giemsa stain is then poured off and the plates are dippedthree times in cold tap water in a beaker. The plates are then inverted,shaken vigorously, and air dried overnight (with plate lids off) on arack on a laboratory bench. Cells per well are then counted manually orby automated and/or computerized means, to derive data regardingchemosensitivity of cells at various concentrations of exposure. Oneparticularly useful computer operating environment for counting cells isthe commercially available OPTIMATE compiler, which is designed topermit an optical counting function well suited to computerized cellcounting procedures and subsequent calculations.

The above procedures do not change appreciably when cell growthpromoters are assayed rather than cell arresting agents such aschemotherapeutic agents. The present assay allows cell death or cellgrowth to be monitored with equal ease. In any case, optimization of useof the present system will involve the comparative testing of a varietyof candidate active agents for selection of the best candidate forpatient treatment based upon the in vitro results. One particularlyadvantageous embodiment of the above described invention comprises atwo-stage assay for cytotoxicity followed by evaluation of longer-terminhibitory effect. Chemotherapeutic agents may thus be evaluatedseparately for both their direct chemotherapeutic effect as well as fortheir longer duration efficacy.

Identification of one or more active agents or chemotherapeutic agentsis peripheral to the present invention, which is intended for theefficacy screening of any or all of them as to a given patient.Literally any active agent may be screened according to the presentinvention; listing exemplary active agents is thus omitted here.

The essence of the invention thus includes the important feature of thesimplicity of the present system-cohesive multicellular particulates ofthe patient tissue to be tested are used to form cell monolayers; growthof those monolayers is monitored for accurate prediction of correlatinggrowth of the same cells in vivo; and differing concentrations of anumber of active agents may be tested for the purpose of determining notonly the most appropriate agent but the most appropriate concentrationof that agent for actual patient exposure (according to the calculatedcell growth rates). It is also important to note, in the context of theinvention, that the present system allows in vitro tests to be conductedin suspensions of tissue culture monolayers grown in nutrient mediumunder fast conditions (a matter of weeks), rather than with single cellprogeny produced by dilution cloning over long periods of time. In somecases, the present invention is a two stage assay for both cytotoxicityand the longer-term growth inhibitory.

Another important aspect of the present invention is to provide a systemfor screening specific tissue samples from individual patients forexpressed cellular markers, secreted factors or antigens, includingtumor antigens, characteristic of the tissue sample. A tissue samplefrom a patient is harvested and grown in a monolayer culture asdescribed above. Culture medium in which the primary monolayer cultureor subcultures thereof can then be assayed for the presence or absenceof certain factors, such as secreted tumor antigens like PAI-1, u-PA orcarcinoembryonic antigen. These factors may be detected through use ofstandard assays such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA) although the many other assays known tothose skilled in the art may be used to detect and/or quantify thesoluble factors. The cell cultures grown in this manner may also beassayed histochemically and or immunohistochemically for identificationor quantification of cellular or membrane-bound markers. By screeningtissue samples in this manner for production of such factors, markers orantigens, the cultured cells may be further identified, aiding thephysician in treatment strategies and as a prognosis indicator.Furthermore, by combining the use of the culture technique with assayingfor such markers, factors and antigens, a treatment strategy for adisease state may be optimized and treatment progression may bemonitored.

Lastly, immunological markers may be monitored in applications requiringup- or down-regulation of such markers (i.e., Major histocompatibilitycomplex molecules). This aspect of the present invention can beespecially useful in transplantation applications where, for instance,through chemical or biological means rejection of transplanted cells issought to be avoided by down-regulation of the various transplantationantigens present on the cells to be transplanted. The present inventionwould be especially useful in monitoring such immunoregulation.

EXAMPLE 1 Radiation Therapy

Separate 50 mg samples from residual tissue from specimens of threehuman glioblastomas and one human ovarian carcinoma were minced inmedium with sterile scissors to a particle size of roughly 1 mm³ andwith a particle size distribution between about 0.25 and about 1.5 mm3.The medium was Standard F-10 medium containing 17% calf serum and astandard amount of Penicillin and Streptomycin. Each 50 mg sample wasminced and was divided into four groups of particulates and each of 16groups was charged to a separate labeled culture flask containing theabove-described medium. Visual confirmation was made that theparticulates were evenly distributed along the bottom of each flask andthe flasks were placed in a 35° C., non-CO₂ incubator. Flasks werechecked daily for growth and contamination. Over a period of a fewweeks, with weekly removal and replacement of 5 ml of growth medium, theparticulates grew into monolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the 16flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification—only those cells whichhad not taken up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated and by means known in the art the optimumconcentration of cells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the16 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours later, the cells were irradiated using a SiemensStabilipan X-ray machine at 250 kVp, 15 mA with a dose rate of 75rad/minute. For each radiation dose from 1Gy to 6Gy, cell number perwell was monitored as a function of time through five dayspost-irradiation.

Cell number relative to controls was determined and survival curves werefit to the data. The rate of decrease in survival as a function of timewas proportional to dose. A differential radiation response among thefour cell lines was observed.

EXAMPLE 2 Immuno Therapy

Separate 50 mg samples from residual tissue from specimens of a humanbrain tumor, renal carcinoma, and breast carcinoma were minced in mediumwith sterile scissors to a particle size of roughly 1 mm³ and with aparticle size distribution between about 0.25 and about 1.5 mm³. Themedium was Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin. Each 50 mg sample was minced andwas divided into four groups of particulates and each of 12 groups wascharged to a separate labeled culture flask containing theabove-described medium. Visual confirmation was made that theparticulates were evenly distributed along the bottom of each flask andthe flasks were placed in a 35° C., non-CO₂ incubator. Flasks werechecked daily for growth and contamination. Over a period of a fewweeks, with weekly removal and replacement of 5 ml of growth medium, theparticulates grew into monolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of thetwelve flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well a 96-well microtiter plate together with an equal amountof trypan blue, plus stirring. The resulting admixture was then dividedbetween two hemocytometer quadrants for examination using a standardlight microscope. Cells were counted in two out of four hemocytometerquadrants, under 10× magnification—only those cells which had not takenup the trypan blue dye were counted. This process was repeated for thesecond counting chamber. An average cell count per chamber wascalculated and by means known in the art the optimum concentration ofcells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the12 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours post-plating, Activated Natural Killer (ANK)cells were delivered into a row of six wells by means of a micropipette.In each microtiter plate three rows of six wells each served ascontrols. The effector (ANK cells): target cell (tumor cells) ratiovaried from 2.5:1 to 20:1. The ANK cells were exposed to the targetcells for four hours. Subsequently, the wells were washed with HanksBalanced Salt Solution and the number of ANK cells remaining in thewells was observed with a phase contrast microscope. This process wasrepeated until no ANK cells remained in the wells (usually 3 washes).Following removal of the ANK cells, the tumor cells were incubated inthe wells for another 24 hours.

Cell number relative to control was determined. For the three tumortypes increasing the effector:target cell ratio from 2.5:1 to 20:1resulted in an increase in the number of tumor cells killed by the ANKcells.

EXAMPLE 3 Gene Therapy/Antisense Oligonucleotides

A 50 mg sample from a residual human mesothelioma was minced in mediumwith sterile scissors to a particle size of roughly 1 mm³ and with aparticle size distribution between about 0.25 and about 1.5 mm³. Themedium was Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin. The 50 mg sample was minced andwas divided into four groups of particulates and each of four groups wascharged to a separate labeled culture flask containing theabove-described medium. Visual confirmation was made that theparticulates were evenly distributed along the bottom of each flask andthe flasks were placed in a 35° C., non-CO₂ incubator. Flasks werechecked daily for growth and contamination. Over a period of a fewweeks, with weekly removal and replacement of 5 ml of growth medium, theparticulates grew into monolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the fourflasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification—only those cells whichhad not taken up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated and by means known in the art the optimumconcentration of cells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from thefour monolayers were separately suspended in growth medium via vortexand rocking and were loaded into a Terasaki dispenser adapted to a60-well plate. Aliquots of the prepared cell suspension were deliveredinto the microtiter plates using Terasaki dispenser techniques known inthe art. Cells were plated into 60-well microtiter plates at aconcentration of 100 cells per well.

Twenty-four (24) hours post-plating, antisense oligonucleotide for theurokinase-type plasminogen activator receptor (uPAR) was delivered towells in the microtiter plate. Proteolysis of plasminogen to plasmin byurokinase-type plasminogen activator has been implicated in theprocesses of tumor cell proliferation and invasion. The concentrationsof the uPAR antisense oligonucleotide were 1, 10 and 100 micromolar.uPAR sense and missense oligonucleotides at the concentrations of 1, 10and 100 micromolar served as controls. The tumor cells were exposed tothe oligonucleotides for 24 hours and then the agents were removed. Thecells were allowed to incubate for another 72 hours so that inhibitionof cell proliferation could be observed.

Cell number relative to control was then determined. Antisenseoligonucleotides to uPAR suppressed the proliferative activity of thetumor cells in a concentration dependent manner.

EXAMPLE 4 Combination Chemotherapy

Separate 50 mg samples from residual tissue from specimens from fourhuman ovarian tumors were minced in medium with sterile scissors to aparticle size of roughly 1 mm³ and with a particle size distributionbetween about 0.25 and about 1.5 mm³. The medium was Standard F-10medium containing 17′ calf serum and a standard amount of Penicillin andStreptomycin. Each 50 mg sample was minced and was divided into fourgroups of particulates and each of 16 groups was charged to a separatelabeled culture flask containing the above-described medium. Visualconfirmation was made that the particulates were evenly distributedalong the bottom of each flask and the flasks were placed in a 35° C.,non-CO₂ incubator. Flasks were checked daily for growth andcontamination. Over a period of a few weeks, with weekly removal andreplacement of 5 ml of growth medium, the particulates grew intomonolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the 16flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well a 96-well microtiter plate together with an equal amountof trypan blue, plus stirring. The resulting admixture was then dividedbetween two hemocytometer quadrants for examination using a standardlight microscope. Cells were counted in two out of four hemocytometerquadrants, under 10× magnification—only those cells which had not takenup the trypan blue dye were counted. This process was repeated for thesecond counting chamber. An average cell count per chamber wascalculated and by means known in the art the optimum concentration ofcells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the16 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours post-plating, the chemotherapeutic agent taxolwas applied to the wells in the microtiter plates. The first threetreatment rows in the plates (Rows 2, 3, and 4) were designed to haveescalating taxol doses (1.0, 5.0, and 25 μM) with a fixed carboplatindose (200 μM). The last three treatment rows in the plates (Rows 6, 7,and 9) were designed to have a fixed taxol dose (5 μM) with anescalating carboplatin dose (50, 200, and 1000 μM). Rows 5 and 9 servedas a control. The taxol exposure time was two hours. Twenty-four hourslater, the cells in the wells were exposed to carboplatin for two hours.The tumor cells in the wells were then incubated for another 48 hours.

Cell number relative to control was determined. For the cells from thefour tumor specimens a dose response relationship was observed for boththe escalating taxol/fixed carboplatin and fixed taxol/escalatingcarboplatin treatment schema.

EXAMPLE 5 Hormonal Therapy

Separate 50 mg samples from residual tissue from specimens from fourhuman breast tumors were minced in medium with sterile scissors to aparticle size of roughly 1 mm³ and with a particle size distributionbetween about 0.25 and about 1.5 mm³. The medium was Standard F-10medium containing 17% calf serum and a standard amount of Penicillin andStreptomycin. Each 50 mg sample was minced and was divided into fourgroups of particulates and each of 16 groups was charged to a separatelabeled culture flask containing the above-described medium. Visualconfirmation was made that the particulates were evenly distributedalong the bottom of each flask and the flasks were placed in a 35° C.,non-CO₂ incubator. Flasks were checked daily for growth andcontamination. Over a period of a few weeks, with weekly removal andreplacement of 5 ml of growth medium, the particulates grew intomonolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the 16flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification—only those cells whichhad not taken up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated and by means known in the art the optimumconcentration of cells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the16 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours post-plating, the antiestrogenic compoundtamoxifen was delivered to wells in the microtiter plates. A stocksolution of tamoxifen was initially prepared by dissolving 1.5 mg oftamoxifen powder in 1 ml of absolute ethanol and then adding 9 ml ofgrowth medium. This stock solution was then used to make tamoxifensolutions in the concentration range of 10 nM to 20 μM. Six doses oftamoxifen were used for cells from each of the four breast tumorspecimens. An ethanol solution at a concentration equivalent to that atthe highest tamoxifen concentration served as a control. The tumor cellswere exposed to tamoxifen for 24 hours and then the agent was removed.The cells were allowed to incubate for another 72 hours so thatinhibition of cell proliferation could be observed.

Cell number relative to control was then determined. There was no effectobserved when the ethanol-only control wells were compared to the growthmedium-only control wells. The cells of two of the four breast specimenstested showed an inhibition of cell proliferation by tamoxifen exposure.These responses occurred in the mid to high tamoxifen concentrationranges.

EXAMPLE 6 Differentiating Agent Therapy (“Biological ResponseModification”)

Separate. 50 mg samples from residual tissue from specimens from fourhuman breast tumors were minced in medium with sterile scissors to aparticle size of roughly 1 mm³ and with a particle size distributionbetween about 0.25 and about 1.5 mm³. The medium was Standard F-10medium containing 17% calf serum and a standard amount of Penicillin andStreptomycin. Each 50 mg sample was minced and was divided into fourgroups of particulates and each of 16 groups was charged to a separatelabeled culture flask containing the above-described medium. Visualconfirmation was made that the particulates were evenly distributedalong the bottom of each flask and the flasks were placed in a 35° C.,non-CO₂ incubator. Flasks were checked daily for growth andcontamination. Over a period of a few weeks, with weekly removal andreplacement of 5 ml of growth medium, the particulates grew intomonolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the 16flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification only those cells whichhad not taken up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated and by means known in the art the optimumconcentration of cells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the16 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours post-plating the differentiating agent retinoicacid was delivered to wells in the microtiter plates. A stock solutionof retinoic acid was initially prepared by dissolving retinoic acidpowder in 1 ml of dimethyl sulfoxide (DMSO) and then adding 9 ml ofgrowth medium. This stock solution was then used to make retinoic acidsolutions in the concentration range of 0.1 to 1.0 mM. Six doses ofretinoic acid were used for cells from each of the four breast tumorspecimens. A DMSO solution at a concentration equivalent to that at thehighest retinoic acid concentration served as a control. The tumor cellswere exposed to retinoic acid for 24 hours and then the agent wasremoved. The cells were allowed to incubate for another 72 hours so thatinhibition of cell proliferation could be observed.

Cell number relative to control was then determined. There was no effectobserved when the DMSO-only control wells were compared to the growthmedium-only control wells. The cells of three of the four breastspecimens tested showed an inhibition of cell proliferation by retinoicacid exposure. These responses occurred in the mid to high retinoic acidconcentration ranges.

EXAMPLE 7 Combined Modality Therapy Drug/Radiation

Separate 50 mg samples from residual tissue from specimens from twohuman brain tumors and two human ovarian tumors were minced in mediumwith sterile scissors to a particle size of roughly 1 mm³ and with aparticle size distribution between about 0.25 and about 1.5 mm³. Themedium was Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin. Each 50 mg sample was minced andwas divided into four groups of particulates and each of 16 groups wascharged to a separate labeled culture flask containing theabove-described medium. Visual confirmation was made that theparticulates were evenly distributed along the bottom of each flask andthe flasks were placed in a 35° C., non-CO₂ incubator. Flasks werechecked daily for growth and contamination. Over a period of a fewweeks, with weekly removal and replacement of 5 ml of growth medium, theparticulates grew into monolayers.

Enough cells were then removed from the monolayers grown in the flasksfor centrifugation into standard size cell pellets for each of the 16flasks. Each cell pellet was then suspended in 5 ml of theabove-described medium and was mixed in a conical tube with a vortex for6 to 10 seconds, followed by manual rocking back and forth 10 times. A36 microliter droplet from the center of each tube was then pipettedinto one well of a 96-well microtiter plate together with an equalamount of trypan blue, plus stirring. The resulting admixture was thendivided between two hemocytometer quadrants for examination using astandard light microscope. Cells were counted in two out of fourhemocytometer quadrants, under 10× magnification only those cells whichhad not taken up the trypan blue dye were counted. This process wasrepeated for the second counting chamber. An average cell count perchamber was calculated and by means known in the art the optimumconcentration of cells in the medium was determined.

Accommodating the above calculations, additional cell aliquots from the16 monolayers were separately suspended in growth medium via vortex androcking and were loaded into a Terasaki dispenser adapted to a 60-wellplate. Aliquots of the prepared cell suspension were delivered into themicrotiter plates using Terasaki dispenser techniques known in the art.Cells were plated into 60-well microtiter plates at a concentration of100 cells per well.

Twenty-four (24) hours post-plating, cells in the microtiter plate wellswere exposed to the chemotherapeutic agent taxol. One set of plates wasdesigned to have escalating taxol doses with (0.5-25.0 μM) with a fixedradiation dose (2Gy). A second set of plates was designed to have afixed taxol dose (5 μM) with an escalating radiation dose (1Gy-6Gy). Thecells in the plates were irradiated using a Siemans Stabilipan X-raymachine operating at 250 kvp, 15 mA with a dose rate of 75 rad/minute.

For each of the two treatment schema, cell number per well was monitoredas a function of time through 5 days post-treatment. Cell numberrelative to controls was determined and survival curves were fit. Adifferential response among the cells from the four tumor specimens wasobserved. Both additive and synergistic cell killing was noted.

Although the present invention has been described with respect tospecific materials and methods above, the invention is only to beconsidered limited insofar as is set forth in the accompanying claims.

1. A method for preparing a biopsy sample of tissue containing malignantcells for chemosensitivity testing, comprising: (a) obtaining a tumorspecimen containing malignant cells and unwanted cells, wherein saidmalignant cells are susceptible of overgrowth of said unwanted cells;(b) mechanically separating said specimen into cohesive multicellularparticulates having a size of about 0.25-1.25 mm3; (c) growing a tissueculture monolayer from said cohesive multicellular particulates withoutovergrowth of said unwanted cells; (d) inoculating malignant cells fromsaid monolayer into a plurality of segregated sites; (e) treating saidplurality of sites with at least one agent; (f) examining said pluralityof sites; and (g) assessing chemosensitivity of the cells in saidplurality of sites; and (h) monitoring culture medium in which saidmonolayer is grown for production of soluble secreted factors indicativeof a disease state or lack thereof.
 2. The method according to claim 1wherein said agent is at least one agent selected from the groupconsisting of chemotherapeutic agents, radiation therapy agents,radiation therapy sensitizing agents, radiation therapy desensitizingagents, immunotherapeutic agents, gene therapy agents, combinationchemotherapeutic agents, hormone therapy agents, wound healing agents,and differentiating agents.
 3. The method according to claim 1 whereinsaid plurality of segregated sites further comprises a plate containinga plurality of wells therein.
 4. The method according to claim 3 whereinsaid cells in step (d) are prepared in suspension prior to inoculationinto said plurality of wells.
 5. The method according to claim 1 whereinsaid agent is a gene therapy agent and further said gene therapy agentcomprises an antisense oligonucleotide.
 6. The method according to claim1 wherein said soluble secreted factors are cellular markers, secretedfactors or tumor antigens.
 7. The method according to claim 1 whereinsaid soluble secreted factors are selected from the group consisting ofplasminogen activator inhibitors type I, eurokinase-type plasminogenactivator, alpha-fetoprotein, carcinoembryonic antigen, transforminggrowth factor alpha, transforming growth factor beta, and majorhistocompatibility complex.